Real time quantitative PCR is rapidly replacing conventional or "end-point" PCR as a major diagnostic tool. Thermal Gradient Inc. (TG) has already shown that microfluidic devices based on its proprietary technology can carry out end-point PCR quickly and simply for bio-defense applications. "Quickly" is understood to mean reactions completed in minutes instead of hours, as with conventional equipment. TG would like to extend the benefits of its technology into quantitative diagnostic testing. The proposed project, Fast and Simple Real Time PCR for Diagnostic Testing, is intended to establish the feasibility of performing "real time" PCR more rapidly and conveniently than conventional means. The product envisioned here would be a compact instrument that could be used in the operating room or at the bedside to determine, among other applications, the microbial load in body fluids or the extent of tumor invasion in a biopsy, all within 30 minutes of sample collection. All of the technology required to carry this out exists today except for rapid, real-time PCR thermal cycling. In "end-point" PCR for bio-defense, the reaction is carried out to completion and then fluorescent detection is employed to identify from the amplified DNA which among many possible pathogens have been employed in an attack. In "real-time" PCR the objective is to determine from the rate of the reaction the starting concentration of usually one pathogen or a mutant sequence, although detection of multiple targets is also possible. This requires fluorescent detection of the reaction mix while it is undergoing PCR. In this Phase I application, we propose to achieve two aims: 1. Show that a "realtime" version of the TG device, one that permits observing the sample mix as it flows through the device, will still carry out an "end-point" PCR; and 2. Show that it is reasonable to expect that a well designed detection system will be able to detect the fluorescent signal that will be emitted by the "real-time" device. If these goals are met it is very likely that real time PCR can be carried out successfully in a Phase II project meant to demonstrate all the pieces of a very fast, complete, and quantitative molecular diagnostic system. [unreadable] [unreadable] [unreadable]